Download Introduction to pGLO lab

Introduction
to pGLO lab
Bacteria Transformation
QQ#1: What is a plasmid?
A
plasmid is a small circular piece of
DNA (about 2,000 to 10,000 base pairs)
that contains important genetic
information for the growth of bacteria.
How do scientists use plasmids?
A
plasmid are used as vectors.
A
vector is a vehicle for transferring foreign
genetic information
 Often
these plasmids are altered to create
recombinant DNA that contains and
expresses new, foreign traits.
 For
example, our plasmid for this lab contains:
 AmpR:
a gene that codes for resistance to an
antibiotic (ampicillin). Bacteria with these
plasmids can now live in the presence of an
antibiotic.
What do you do with plasmids
that are now recombinant
DNA?
 After
following the steps to combine a bacterial
plasmid with foreign DNA, scientists need to
place the recombinant DNA into a living
organism.
 The recombinant DNA is inserted into a bacteria.
 Then the bacteria will express the new “foreign”
DNA, and the bacteria will perform new
functions.
QQ#2: What is this process called?
Transformation:
The process of inserting
recombinant plasmid DNA into a
bacteria (or any other cell)
How do you know if
Transformation occurred?

The transformed bacteria are then spread over an agar
plate that contains ampicillin.

Because our plasmid contains the ampR gene, only
bacteria that have acquired the plasmid can grow on
the plate.

The ampicillin provides a selective pressure

Selective Pressure - The same as in evolution - only the
organisms that have a particular trait (in this case
antibiotic resistance) will survive.

Therefore, as long as you grow the bacteria in
ampicillin, it will need the plasmid to survive
In the pGLO lab, we will be
performing transformation
 We
will take a plasmid that has been recombined
into a piece of recombinant DNA that contains 3
new genes:



gene from a jellyfish (GFP)
A gene that induces GFP
ampicillin resistance gene.
 When
transformation is complete, and we insert the
plasmid into a bacteria cell, the cell will express the
jellyfish gene and will fluoresce .
Why would we WANT to
make something glow??
http://www.youtube.com/watch?v=Qm5Bj5
xNefM&list=PLNtVKBx_tJzM2jDB8Ow67VV7otRhmSuw
What jellyfish gene
will we use?




GFP is a green fluorescent protein that
normally is found in jellyfish
Osamu Shimomura isolated the GFP from
a jellyfish. Some of Shimomura's
colleagues realized that the protein could
be attached to other proteins--enabling
scientists to mark proteins of their choice
with a green glow.
(GFP) has been used to decrypt previously
invisible processes, like the spread of
cancer or the development of nerve cells-earning Shimomura and colleagues a
Nobel Prize in 2008.
Fluorescent proteins have also been used
to engineer some truly strange beasts
(and the odd plant), such as the glowing
puppies, monkeys, mice, fish and other
animals on the following pages.
Are we going to make kittens glow? No,
just bacteria.
 We
are going to create
transgenic bacteria by
transforming them and
forcing them to take up
a commercially
prepared plasmid that
contains three genes of
interest, ampR, araC
and GFP.
 Remember: Genetically
modified organisms are
“transgenic”
Genes of interest: amp, araC, GFP
 ampR
– this gene will give our transgenic
bacteria resistance to the antibiotic ampicillin
so we know if transformation occurred
 araC
– this gene is induced by arabinose and
creates a protein will induce (switch on) the
GFP gene.
 GFP
– induced with the help of araC this
gene will “switch on” and cause the
transformed (transgenic) bacteria to glow
green
 These
three genes will work together to allow
gene regulation to be on, and for GFP to be
expressed.
QQ#3
 Listen
and draw/follow along with how
your teacher explain pGLO gene
regulation
 Explain
in your own words the relationship
between the three genes of interest.
Our Lab
 Bacterial
 We
Transformation should occur!
will use several different agar plates in
order to see if the transformation was
successful.
How will we transform the bacteria?
1.
2.
3.
4.
5.
Suspend bacterial colonies in Transformation
Solution to weaken the cell wall
Add pGLO plasmid DNA to +DNA tube
Place tubes on ice to make the plasmid stick to
the cell wall
Heat shock at 42oC to force plasmid uptake and
place on ice to close cell wall
Incubate with LB broth to allow nutrients so newly
acquired genes can be expressed.
This process will make the bacteria take up the
plasmid through its cell wall.
We will start with:
 E.
coli starter plate
 This plate has the
bacteria we will use
growing in a luria
broth (LB) agar
plate.
 These bacteria are
normal (have NOT
been transformed)
We will use 4 different petri dish
set ups to see our results
 Each





plate contains different things
LB: luria broth-nutrients
Amp: the antibiotic ampicillin
Ara: the sugar arabinose
+pGLO: contains transformed bacteria
-pGLO: contains non-transformed bacteria
Explanation of agar plates
 LB/-pGLO
 This
is the control plate. These –DNA
bacteria are not transformed and are in
normal (LB) agar.
 You should expect to see normal
bacterial growth in this plate.
Explanation of agar plates
 LB/amp/-pGLO
 These
–pGLO bacteria have not received
the plasmid.
 They have not been transformed, so they
do not have resistance to the ampicillin
that is in the agar.
 There should be no growth on this plateampicillin kills bacteria
Explanation of agar plates
 LB/amp/+pGLO
 This
plate will have E. coli bacteria on LB
agar to which ampicillin has been added.
 The +pGLO means that the bacteria may
have been transformed (if your technique is
good).
 If they have been transformed, they will now
have a plasmid with an ampicillin resistant
site so they will not be killed by the ampicillin
that has been added to the agar.
Explanation of agar plates
 LB/amp/ara/+pGLO
 This
plate will have transformed
bacteria (+pGLO) growing on agar
that has both ampicillin and
arabinose added to it.
 If your technique is good, you should
expect to see green glowing bacteria
in this plate.